T lymphocytes are formed in the bone marrow, migrate to and mature in the thymus and then enter the peripheral blood and lymphatic circulation. T lymphocytes are subdivided into three distinct types of cells: helper T cells, suppressor T cells, and cytotoxic T cells. T lymphocytes, unlike B lymphocytes, do not produce antibody molecules, but express a heterodimeric cell surface receptor that recognizes peptide fragments of antigenic proteins that are attached to proteins of the major histocompatibility complex (MHC) and expressed on the surfaces of target cells; see, e.g., Abbas, A. K., Lichtman, A. H., and Pober, J. S., Cellular and Molecular Immunology, 1991, esp. pages 15-16.
Cytotoxic T lymphocytes (CTLs) are typically of the CD3+, CD8+, CD4- phenotype and lyse cells that display fragments of foreign antigens associated with class I MHC molecules on their cell surfaces. Target cells for CTL recognition include normal cells expressing antigens after infection by viruses or other pathogens; and tumor cells that have undergone transformation and are expressing mutated proteins or are over-expressing normal proteins.
Helper T cells are also CD3+ but can be distinguished from cytolytic T cells by expression of CD4 and absence of the CD8 membrane protein. CD4+ helper T cells recognize fragments of antigens presented in association with class II MHC molecules, and primarily function to produce cytokines that amplify antigen-specific T and B cell responses and activate accessory immune cells such as monocytes or macrophages. See, e.g., Abbas, A. K., et al., supra.
CD4+ helper and CD8+ cytotoxic T lymphocytes are important components of the host immune response to viruses, bacterial pathogens and tumors. As a result, individuals with congenital, acquired or iatrogenic T cell immunodeficiency diseases may develop life threatening infections or malignancies (for example, SCID, BMT, AIDS, etc.). Persons with diseases that are related to a deficiency of immunologically competent T lymphocytes can potentially have specific immunity restored through adoptive immunotherapy, alternatively called adoptive transfer. In adoptive immunotherapy, one or more specific immunities can be conferred upon an individual by transferring T cells having the desired antigenic specificities. The cells of interest may be derived from the immunodeficient host or from a compatible specifically immunized host. The latter source is of course especially important in situations in which the immunodeficient host has an insufficient number of T cells, or has T cells that are insufficiently effective.
In order to augment or reconstitute T cell responses in such immunodeficient hosts, the antigen-specific T cells must be grown to large numbers in vitro and then administered intravenously to the immune-deficient host. After undergoing adoptive immunotherapy, hosts that previously had inadequate or absent responses to antigens expressed by pathogens or tumors, may express sufficient immune responses to become resistant or immune to the pathogen or tumor.
Adoptive transfer of antigen-specific T cells to establish immunity has been demonstrated to be an effective therapy for viral infections and tumors in animal models (reviewed in Greenberg, P. D., Advances in Immunology (1991)). For adoptive immunotherapy to be effective, antigen-specific T cells usually need to be isolated and expanded in numbers by in vitro culture, and following adoptive transfer such cultured T cells must persist and function in vivo. For treatment of human disease, the use in immunotherapy of cloned antigen-specific T cells which represent the progeny of single cells, offers significant advantages because the specificity and function of these cells can be rigorously defined and precise dose:response effects evaluated. Riddell et al. were the first to adoptively transfer human antigen-specific T cell clones to restore deficient immunity in humans. Riddell, S. R. et al., "Restoration of Viral Immunity in Immunodeficient Humans by the Adoptive Transfer of T Cell Clones", Science 257:238-240 (1992). In this study, Riddell et al. used adoptive immunotherapy to restore deficient immunity to cytomegalovirus in allogeneic bone marrow transplant recipients. Cytomegalovirus specific CD8+ cytotoxic T cell clones were isolated from three CMV seropositive bone marrow donors, propagated in vitro for 5 to 12 weeks to achieve numerical expansion of effector T cells, and then administered intravenously to the respective bone marrow transplant (BMT) recipients. The BMT recipients were deficient in CMV-specific immunity due to ablation of host T cell responses by the pretransplant chemoradiotherapy and the delay in recovery of donor immunity commonly observed after allogeneic bone marrow transplant (Reusser et al. Blood, 78:1373-1380, 1991). Riddell et al. found that no toxicity was encountered and that the transferred T cell clones provided these immunodeficient hosts with rapid and persistent reconstitution of CD8+ cytomegalovirus-specific CTL responses.
Riddell et al. (J. Immunology, 146:2795-2804, 1991) used the following procedure for isolating and culturing the CD8+ CMV-specific T cell clones: peripheral blood mononuclear cells (PBMCs) derived from the bone marrow donor were first cultured with autologous cytomegalovirus-infected fibroblasts to activate CMV-specific CTL precursors. Cultured T cells were then restimulated with CMV-infected fibroblasts and the cultures supplemented with .gamma.-irradiated PBMCs. 2-5 U/ml of interleukin-2 (IL-2) in suitable culture media was added on days 2 and 4 after restimulation to promote expansion of CD8+ CTL (Riddell et al., J. Immunol., 146:2795-2804, 1991). To isolate T cell clones, the polyclonal CD8+ CMV-specific T cells were plated at limiting dilution (0.3-0.6 cells/well) in 96-well round bottom wells with either CMV-infected fibroblasts as antigen-presenting cells (Riddell, J. Immunol., 146:2795-2804, 1991); or anti-CD3 monoclonal antibody to mimic the stimulus provided by antigen-presenting cells. (Riddell, J. Imm. Methods, 128:189-201, 1990). Then, .gamma.-irradiated peripheral blood mononuclear cells (PBMC) and EBV-transformed lymphoblastoid cell line (LCL) were added to the microwells as feeder cells. Wells positive for clonal T cell growth were evident in 10-14 days. The clonally derived cells were then propagated to large numbers initially in 48 or 24 inch plates and subsequently in 12-well plates or 75-cm.sup.2 tissue culture flasks. T cell growth was promoted by restimulation every 7-10 days with autologous CMV-infected fibroblasts and .gamma.-irradiated feeder cells consisting of PBMC and LCL, and the addition of 25-50 U/ml of IL-2 at 2 and 4 days after restimulation.
A major problem that exists in the studies described above, and in general in the prior art of culturing T cells, is the inability to grow large quantities of human antigen-specific T cell clones in a timely fashion. It is not known if the slow growth of T cells in culture represents an inherent property of the cell cycle time for human lymphocytes or the culture conditions used. For example, with the culture method used in the CMV adoptive immunotherapy study described above, three months were required to grow T cells to achieve the highest cell dose under study which was 1.times.10.sup.9 T cells/cm.sup.2. This greatly limits the application of adoptive immunotherapy for human viral diseases and cancer since the disease process may progress during the long interval required to isolate and grow the specific T cells to be used in therapy. Based on extrapolation from animal model studies (reviewed in Greenberg, P. D., Advances in Immunology, 1991), it is predicted that in humans doses of antigen-specific T cells in the range of 10.sup.9 -10.sup.10 cells may be required to augment immune responses for therapeutic benefit. However, rapidly expanding antigen-specific human T cells in culture to these cell numbers has proven to be a significant obstacle. Thus, with the exception of the study by Riddell et al., supra, studies of adoptive immunotherapy using antigen-specific T cell clones have not been performed. The problem of producing large numbers of cells for adoptive immunotherapy was identified in U.S. Pat. No. 5,057,423. In this patent, a method for isolating pure large granular lymphocytes and a method for the expansion and conversion of these large granular lymphocytes into lymphokine activated killer (LAK) cells is described. The methods are described as providing high levels of expansion, i.e up to 100-fold in 3-4 days of culture. Although LAK cells will lyse some types of tumor cells, they do not share with MHC-restricted T cells the properties of recognizing defined antigens and they do not provide immunologic memory. Moreover, the methods used to expand LAK cells, which predominantly rely on high concentrations of IL-2 do not efficiently expand antigen-specific human T cells (Riddell et al., unpublished); and those methods can render T cells subject to programmed cell death (i.e. apoptosis) upon withdrawal of IL-2 or subsequent stimulation via the T cell receptor (see the discussion of the papers by Lenardo et al, and Boehme et al., infra).
The inability to culture antigen-specific T cell clones to large numbers has in part been responsible for limiting adoptive immunotherapy studies for human diseases such as cancer (Rosenberg, New Engl. J. Med., 316:890-897, 1987 Rosenberg, New Engl. J. Med., 319:1676-1680, 1988) and HIV infection (Ho M. et al., Blood 81:2093-2101, 1993) to the evaluation of activated polyclonal lymphocyte populations with poorly defined antigen specificities. In such studies, polyclonal populations of lymphocytes are either isolated from the blood or the tumor filtrate and cultured in high concentrations of the T cell growth factor IL-2. In general, these cells have exhibited little if any MHC-restricted specificity for the pathogen or tumor and in the minority of patients that have experienced therapeutic benefit, it has been difficult to discern the effector mechanism involved. Typically, adoptive immunotherapy studies with non-specific effector lymphocytes have administered approximately 2.times.10.sup.10 to 2.times.10.sup.11 cells to the patient. (See, e.g., U.S. Pat. No. 5,057,423, at column 1, lines 40-43).
The development of efficient cell culture methods to rapidly grow T lymphocytes will be useful in both diagnostic and therapeutic applications. In diagnostic applications, the ability to rapidly expand T cells from a patient can be used, for example, to quickly generate sufficient numbers of cells for use in tests to monitor the specificity, activity, or other attributes of a patient's T lymphocytes. Moreover, the capability of rapidly achieving cell doses of 10.sup.9 -10.sup.10 cells will greatly facilitate the applicability of specific adoptive immunotherapy for the treatment of human diseases.
There are several established methods already described for culturing cells for possible therapeutic use including methods to isolate and expand T cell clones. Typical cell culture methods for anchorage dependent cells, (i.e., those cells that require attachment to a substrate for cell proliferation) are limited by the amount of surface area available in culture vessels used (i.e., multi-well plates, petri dishes, and culture flasks). For anchorage dependent cells, the only way to increase the number of cells grown is to use larger vessels with increased surface area and/or use more vessels. However, hemopoietic derived cells such as T lymphocytes are anchorage independent. They can survive and proliferate in response to the appropriate growth factors in a suspension culture without attachment to a substrate. Even with the ability to grow antigen-specific lymphocytes in a suspension culture, the methods reported to date have not consistently produced rapid numerical expansion of T cell clones. For example, in a study of T cells conducted by Gillis and Watson, it was found that T cells cultured at low densities, i.e., 5.times.10.sup.3 to 1.times.10.sup.4 cell/ml in the presence of the T cell growth factor IL-2, proliferated rapidly over a seven day period and eventually reached a saturation density of 3-5.times.10.sup.5 cells/ml. Gillis, S. and Watson, J. "Interleukin-2 Dependent Culture of Cytolytic T Cell Lines", Immunological Rev., 54:81-109 (1981). Furthermore, Gillis and Watson also found that once cells reached this saturation concentration, the cells would invariably die. Gillis et al., id.
Another study reports three different methods for establishing murine T lymphocytes in long-term culture. Paul et al., report that the method most widely used is to grow T lymphocytes from immunized donors for several weeks or more in the presence of antigen and antigen presenting cells to provide the requisite T cell receptor signal and costimulatory signals, and with the addition of exogenous growth factors before attempting to clone them, Paul, W. E., et al., "Long-term growth and cloning of non-transformed lymphocytes", Nature, 294:697-699, (1981). T cells specific for protein antigens are then cloned by limiting dilution with antigen and irradiated spleen cells as a source of antigen-presenting cells (APCs). A second method involves growing T cells as colonies in soft agar as soon as possible after taking the cells from an immunized donor. The T cells are stimulated in an initial suspension culture with antigen and a source of APCs, usually irradiated spleen cells. In this second approach, it has been found that, after 3 days, the cells are distributed in the upper layer of a two-layer soft agar culture system. The colonies may be picked from day 4 to 8 and then expanded in long-term cultures. The third approach involves selecting cells for their functional properties rather than their antigenic specificity and then growing them with a series of different irradiated feeder cells and growth factor containing supernatants. Paul, W. E. et al., "Long-term growth and cloning of non-transformed lymphocytes", Nature, 294:697-699, (1981). It is apparent that with each of these methods, it is not possible to expand individual T cell clones from a single cell to 10.sup.9 -10.sup.10 cells in a timely manner. Thus, despite the ability to clone antigen-specific T cells, and convincing evidence of the therapeutic efficacy of T cell clones in accepted animal models, the technical difficulty in culturing human T cells to large numbers has impeded the clinical evaluation of specific adoptive immunotherapy.
Yet another concern with cultured T cells is that they must remain capable of functioning in vivo in order to be useful in adoptive immunotherapy. In particular, it has been observed that antigen-specific T cells which were grown long term in culture in high concentrations of IL-2 may develop cell cycle abnormalities and lose the ability to return to a quiescent phase when IL-2 is withdrawn. In contrast, the normal cell cycle consists of four successive phases: mitosis (or "M" phase) and three phases which make up the "interphase" stage. During the M phase, the cell undergoes nuclear division and cytokinesis, which is cytoplasmic division. The interphase stage consists of the G.sub.1 phase in which the biosynthetic activities resume at a high rate after mitosis; the S phase in which DNA synthesis begins and continues until the DNA content of the nucleus has doubled and the chromosomes are replicated; and the G.sub.2 phase which continues until mitosis commences. While in the G.sub.1 phase, some cells appear to cease progressing through the division cycle; and are said to be in a "resting" or quiescent state denoted as the "G.sub.0 " state. Certain environmental factors (such as a lack of growth factors in serum or confluence of cell cultures) may cause cells to enter the quiescent state. Once the factor is restored, the cell should resume its normal progress through the cycle. However, cells grown in culture may be unable to enter the quiescent phase when the growth factor is removed, resulting in the death of these cells. This growth factor dependence is particularly relevant to cultured T cells. T lymphocytes that are exposed to high concentrations of IL-2 to promote cell growth often will die by a process called apoptosis if IL-2 is removed or if they are subsequently stimulated through the T cell receptor, i.e., if they encounter specific antigens. (Lenardo M. J., Nature, 353:858-861, 1991; Boehme S. A. and Lenardo M. J., Eur. J. Immunol., 23:1552-1560, 1992). Therefore, the culture methods used to propagate LAK cells or TIL-cells and prior methods to culture T cells which predominantly rely on high concentrations of IL-2 to promote expansion in vitro may render many of the cells susceptible to apoptosis, thus limiting or eliminating their usefulness for adoptive transfer.
It may also be advantageous in adoptive immunotherapy studies to use gene transfer methods to insert foreign DNA into the T cells to provide a genetic marker, which facilitates evaluation of in vivo migration and survival of transferred cells or to confer functions that may improve the safety and efficacy of transferred T cells. An established method for stable gene transfer into mammalian cells is the use of amphotropic retroviral vectors (Miller A D, Current Topics in Microbiology and Immunology, 158:1-24, 1992). The stable integration of genes into the target cell with retrovirus vectors requires that the cell be actively cycling, specifically that these cells transit M phase of the cell cycle. Prior studies have introduced a marker gene into a small proportion of polyclonal T cells driven to proliferate with high doses of IL-2 and these cells were reinfused into humans as tumor therapy and provided a means of following the in vivo survival of transferred cells. (Rosenberg et al. New Engl. J. Med., 323:570-578, 1990). However, for human T cells (which cycle slowly when grown with standard techniques) the efficiency of stable gene transfer is very low, in the range of 0.1-1% of T cells. (Springett C M et al. J. Virology, 63:3865-69,1989). Culture methods which more efficiently recruit the target T cells into the S and G2-M phases of the cell cycle may increase the efficiency of gene modification using retrovirus-mediated gene transfer (Roe T. et al., EMBO J, 2:2099-2108, 1993), thus improving the prospects for using genetically modified T cells in adoptive immunotherapy or using T cells to deliver defective genes in genetic deficiency diseases.